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Fig. 3. iFLAP imaging. (A) Schematic representation of the iFLAP imaging approach. (Left) Using the 514 nm laser of the confocal laser-scanning microscope the YFP part of STAT3-CY can be selectively bleached. (Right) The images of STAT-CY in the YFP and CFP channels of the confocal microscope (original data) are processed into zero signal intensity depicted as blue (intensity= 1–(iYFP/iCFP). Selective bleaching of YFP within the rectangular bleaching-ROI (ROI=region of interest) generates a subpopulation of STAT3-CY molecules that are only detected in the CFP-channel. These molecules generate a signal in the processed data channel. They diffuse freely through the cytoplasm and finally appear within the nucleus. (B) HepG2 transiently transfected with STAT3-CY were analyzed in a perfusion chamber for live cell imaging at the confocal laser-scanning microscope. Two neighbouring cells were selected for imaging (upper-left image shows the merged YFP and CFP channels). The YFP and CFP fluorescence along the red line are shown in the upper-right diagram as yellow and blue graphs, respectively (N, fluorescence intensity within the nucleus). The lower cell was selected for the iFLAP experiment. A bleaching-ROI (white rectangle) was placed into its cytoplasmic compartment, the bleaching procedure was performed, and fluorescence intensity signals of both CFP and YFP were recorded every 5 minutes. Detected signals were recalculated by using the algorithm described in A to generate a signal that represents the distribution of the bleached subpopulation of STAT3-CY over time. As a control the second cell was monitored without bleaching. Relative fluorescence is depicted in false colour mode corresponding to its intensities. Fluorescence intensity profiling (red arrows and right diagrams) is depicted to show the increasing signal in the nuclear compartment. (C) The same experiment was performed as described in B with HepG2 cells coexpressing STAT3-CY and v-Src. (D) The same experiment was performed as described in C in the presence of 100 ng/ml ratjadone A.
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